Effects of hypoxia and ASIC3 on nucleus pulposus cells: From cell behavior to molecular mechanism.

Abstract

This study aimed to explore the effects of hypoxia and acid-sensing ion channel 3 (ASIC3) on nucleus pulposus cells from cell behavior to molecular mechanism. Primary rabbit nucleus pulposus cells were isolated and identified by HE, toluidine blue and immunohistochemical staining of collagen II. 2% O2 and 48 h were screened as optimal oxygen concentration and effect time, respectively, by determining cell apoptosis and mRNA expression of ASIC3, hypoxia inducible factor-1α (HIF-1α) and aquaporin 3. FLuo-3 AM labeling showed that the Ca2+ concentration in cells increased under hypoxia condition. shRNA-ASIC3 and ASIC3 expression vector were transfected into cells. Subsequently, cells were divided into six groups: Control, 2% O2, shRNA-NC+2% O2, shRNA-ASIC3 + 2% O2, Vector+2% O2 and ASIC3 + 2% O2. Flow cytometry, CCK-8 assay, transmission electron microscopy, immunofluorescent labeling, RT-PCR and western blot demonstrated that hypoxia and ASIC3 over-expression inhibited the proliferation, arrested cell cycle in G1 phase, promoted the apoptosis, initiated the autophagy and up-regulated the expression of ASIC3, HIF-1α, light chain 3, p-ERK1/2 and p-MAPK. However, ASIC3 silencing could significantly relieve these phenomena. Co-immunoprecipitation assay found ASIC3 was interacted with HIF-1α&ERK1/2. Evaluation of the effect of HIF-1αsilencing on ASIC3 expression showed that the high expression of ASIC3 induced by hypoxia was reduced significantly by HIF-1α silencing. In conclusion, hypoxia and ASIC3 changed the behavior of nucleus pulposus cells by activating the MAPK pathway. HIF-1α and ASIC3 could regulate each other in nucleus pulposus cells.

• Authors: Dong Wang, Hang Zhu, Wei Cheng, Shiming Lin, Rongxue Shao, Hao Pan
• Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie (Biomed Pharmacother) Vol. 117 Pg. 109061 (Sep 2019) ISSN: 1950-6007 [Electronic] France
• PMID: 31202172 (Publication Type: Journal Article)
• Copyright: Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.

Chemical References

• Acid Sensing Ion Channels
• Hypoxia-Inducible Factor 1, alpha Subunit
• RNA, Messenger
• Oxygen
• Calcium

Topics

• Acid Sensing Ion Channels (genetics, metabolism)
• Animals
• Apoptosis (drug effects)
• Autophagosomes (metabolism, ultrastructure)
• Calcium (metabolism)
• Cell Cycle
• Cell Hypoxia
• Cell Survival
• Cells, Cultured
• Hypoxia-Inducible Factor 1, alpha Subunit (genetics, metabolism)
• MAP Kinase Signaling System
• Nucleus Pulposus (pathology, ultrastructure)
• Oxygen (pharmacology)
• Phosphorylation
• Protein Binding
• RNA, Messenger (genetics, metabolism)
• Rabbits
• Time Factors